Process for the large scale production of human growth hormone by serial secondary suspension culture

ABSTRACT

A new system of serially culturing human anterior pituitary gland cells in a new nutrient medium to produce large amounts of human growth hormone. Since only human growth hormone can be used to treat growth deficiencies in man, there is a great demand for the hormone, which is in relative short supply since only 2 to 3 mgs. of the human growth hormone can be extracted from one human pituitary gland obtained at autopsy. Using the new system of culture and new nutrient media, the human pituitary cells can be grown in vitro to produce in approximately three weeks, more than 20 times the amount of extractable growth hormone than that which was originally present in the original tissue now used for extracting the 2 to 3 mgs. of the hormone.

BACKGROUND OF THE INVENTION

This invention pertains to the field of medicine and medical biology.The specific sub-area of medicine and medical biology is the art of thein vitro culture of mammalian tissue cells. Many nutrient culturemediums have been developed and are, to a large extent, specificallydesigned to permit prolonged and sustained growth of specific types ofcells, such as those derived from skin, from tumors, and from variousother tissues in mammalian species. These are grown by primary culturesystems as explants, monolayers or suspension cultures or secondary celllines. However, there is no culture system or nutrient media availableat the present which will permit prolonged growth and proliferation ofhuman anterior pituitary growth hormone producing cells in sufficientnumber to provide a source of large amounts of growth hormone. This lackof success is related to many factors, among which are (1) limitedknowledge concerning the specific nutrients for the specific cells, (2)the general trend for the pituitary growth hormone-producing cells tolose their function of producing growth hormone, (3) death of cellsafter a few days to a few weeks, and (4) transformation of cells tonon-functioning fibroblastic or fibroblastoid type cells.

Some of the previous studies which indicate the difficulties encounteredare as follows: Animal and human pituitary cells have been maintained asexplants in cultures and are known to release their hormones forvariable periods of time. Continuous cell lines derived from inducedtumors in rats may proliferate for several weeks or a few months and areable to secrete some amounts of rat growth hormone. (Takemoto, H.,Yokoro, K., Furth, J. and Cohen, A. I., Cancer Res., 22:917-924, 1962;Tashjian, A. H. and Hoyt, R. F., Jr., In Molecular Genetics andDevelopmental Biology, edited by M. Sussman, pp. 353-387, Prentice-Hall,New Jersey, 1972). Tumors of the human pituitary gland have also beenmaintained in culture and may secrete human growth hormone into theculture media for variable periods of time (Kohler, P. O., Bridson, W.E., Rayford, P. L. and Kohler, S. E., Metabolism, 18:782, 1969;Batzdorf, U., Gold, V., Matthews, N. and Brown, J., Neurosurgery 34:741,1971; Peillon, F., Gourmelen, M., Donnadieu, M., Brandi, A., Sevaux, D.and Pham Huu Trung, M. T., Acta Endocrinol., 79:217-229, 1975) whereasnormal human pituitary cells in culture survive and produce hormone onlyfor a relatively brief period of time. The number of cells and theamount of hormone produced in such systems decline rapidly with time(Vidal-Tixier, A., Gourdji, D. and Tougard, C., Intern. Rev. Cytol.,41:173-239, 1975). Human fetal pituitary cells secreting growth hormonehave also been cultured for several days but ultimately die or aretransformed into fibroblast type cells (Gailani, S. D., Nussbaum, A.,McDougall, W. J. and McLimans, W. F., Proc. Soc. Exp. Biol. and Med.,134:27-32, 1970).

The above described previously reported results by others were obtainedby a culture procedure in which either small explanted pieces of tissueor a monolayer of cells were grown in small volumes in medium (Peillonet al, Vidal-Tixier et al and Gailani et al, cited above), into whichonly a small amount of growth hormone was released or secreted by thecells. The nutrient mediums used in these cultures have been unable topermit replication of the growth hormone producing cells. In all ofthese previously reported studies, the rate of cell multiplication andthe amount of hormone produced, while persisting for a brief period oftime, rather rapidly declines (Peillon et al). The culture systems ormedia used did not adequately meet the growth and metabolic needs of thecells as conversion to fibroblastoid cells, which are nonhormoneproducing or a loss of the growth hormone synthesis capacity in thecells or death of the cells occurred.

The literature concerning the art of tissue culture procedure and ofnutrient medium developed is very voluminous and particularly extensiveover the last 20 to 25 years. The inventors have, to date, been unableto find evidence of a previously described culture system or nutrientmedium which permits the growth and proliferation of human growthhormone producing cells in sufficient amounts and over a long period oftime to produce an adequate amount of growth hormone which can bebiochemically extracted and used to treat growth hormone deficientstates in man.

SUMMARY OF THE INVENTION

Broadly stated, the invention comprises a process involving a newculture system and new culture media for growing in vitro normal humanpituitary cells for producing large amounts of human growth hormone.More specifically, the invention comprises a process for producing humangrowth hormone in which cells of the human anterior pituitary gland aredispersed in an amino acid-rich nutrient medium supplemented with liverextract, insulin and anti-biotic and anti-fungal agents and incubatedunder open-aeration cell growth conditions. The resulting culture isserially sub-cultured several times until the optimum desired cellgrowth level is achieved. The human growth hormone is extracted .[.fromthe cells.]. using conventional techniques.

DETAILED DESCRIPTION OF THE INVENTION

An exemplary nutrient medium rich in amino acids is that known as Medium199-1X with Earle's modified salts (Proc. Soc. Exp. Biol. Med., 73:1,1950, Growth, 15:11, 1951, Proc. Soc. Exp. Biol. Med., 74:22, 1950;Proc. Soc. Exp. Biol. Med., 78:880, 1951; J. Cell & Comp. Physiol.,36:411, 1950; J. Am. Med. Assn., 151:1081, 1953). To each liter ofmedium there is added from about 5 to 20 ml of liver extract, preferablyabout 10 ml; about 6 to 20 International Units of insulin, preferablyabout 10 I.U.; and minor amounts of anti-biotic and anti-fungal agents.

A preferred nutrient composition according to the present invention isas follows:

(1) Medium 199-1X with Earle's Modified Salts, GIBCO (Grand IslandBiological Company), 1000 ml.

(2) Liver extract containing B vitamins (Lexavite Injectable, Eli Lillyand Company), 10 ml.

(3) Insulin, Crystalline, 10 International Units.

(4) Crystalline Sodium Penicillin G, 500,000 units.

(5) Streptomycin Sulfate, 500 mg.

(6) Nystatin, a polyene anti-fungal anti-biotic, 50,000 units.

The insulin should not be added to the medium until time of use becauseinsulin will degrade in the medium between 4 and 8 days, particularlywhen kept at room temperature or above.

To achieve sustained growth and proliferation of human anteriorpituitary cells and obtain a large cell mass to extract large amounts ofhuman growth hormone, a novel modified system of culturing is used. Thisinvolves the growing of dispersed cells of the human anterior pituitaryin containers containing our new medium with an open-aeration system fora period of several days, preferably about 8 days, followed by serialsub-culturing about every 4 to 8 days. The cultures may be grown asstill-suspension cultures or as rotary suspension cultures. Thetechnique involves greater degrees of aeration than is presentlyemployed in culturing of human and mammalian cells. This is achieved byhaving open air vent caps on the containers which are placed in a 37° C.incubator with water troughs for moisture, provided with an airflowsystem of about 8 to 16 liters of sterile air per minute. Addition of 5%CO₂ to the air-flow which is usually employed in presently availabletissue culture techniques is avoided since we have found that humanpituitary cells do not grow well in the presence of 5% CO₂.

Following about 8 days growth the cultures are sub-cultured serially,utilizing an appropriate dilution range of 1 volume of medium containingcells added to 1 volume of fresh medium. The original culture may bedivided into 2, 3 or more parts, depending upon the quantitative cellcount found at each sub-culture time. If cell numbers are large bymicroscopic examination, greater sub-culture, dilution transfers shouldbe employed. If cells are fewer in number, less dilution transfers ofmedium containing cells to fresh medium should be used. Cultures shouldbe protected from bacterial, fungal and viral contamination at alltimes.

The invention is further illustrated by the following example: Using thepreferred nutrient composition described above, an initial tissueinoculum (about 1/3 of an anterior pituitary gland) was grown in serialculture with 1/2 volume transfers every 8 days for 32 days. Thefollowing average concentrations of hGH in ng/ml found in the mediumwere: at 8 days 21,166; at 16 days 18,666; at 24 days 21,750; and at 32days 20,000. Each culture flask contained 30 ml and hence because ofone-half dilutions and volume reconstitution at sub-culture times, thetotal amount of hGH produced at each sub-culture time was as follows: 8days 5.01 mg in 6 flasks; at 16 days 6.7 mg in 12 flasks; at 24 days15.6 mg in 24 flasks; and, by 32 days 28 mg in 48 flasks. Cultures havebeen grown for as long as two months. Preliminary characterizationstudies utilizing a Sephadex gel filtration procedure indicate that 80to 90% of the immune reacting hGH is a monomer. Cells were epithelialtype, 10-14μ in diameter, contained chromatin granules and a largehighly chromatic nucleus. Mitosis was present.

By this new technique of serial sub-culture, one is able to obtain alarge mass of human pituitary cells for producing large amounts of humangrowth hormone, in relatively short periods of time.

The effectiveness of the medium resides in the addition of certain ofthe medium components which have not heretofore been utilized. Themedium avoids the early death of cells as compared with previous culturemediums that have been used. The pituitary cells are able to incorporateradioactive labeled thymidine which is an evidence of cell growth andproliferation. The medium also prevents the growth or formation offibroblasts or the conversion of the anterior pituitary growth hormonecells into a fibroblastic type cell. Fibroblastic transformation is acommon problem with presently available tissue culture systems. Themedium permits cell growth and multiplication as is evident by mitoticactivity within the cells, and a two to four-fold growth about every 8days of cells which continue to be capable of continuing production ofgrowth hormone. This has been repeatedly confirmed by radioimmune assayfor human growth hormone at every sub-culture done to increase thenumber of flasks with cells producing growth hormone. We have found thathuman growth hormone undergoes a degradation of 50% every three days inthe medium used for their growth and proliferation. In spite of such aconcommitant degradation, we have been able to obtain large amounts ofhuman growth hormone by this new culture technique utilizing our newmedia.

There is a great need to produce human anterior pituitary growth hormoneas it is, at present, in high demand. At present human growth hormone isprepared by a process of direct extraction of growth hormone from freshpituitary glands obtained from humans at autopsy. The amount of humangrowth hormone that can be produced by this method is very small. Thesupply now available is but a fraction of the amount needed to treathuman infants and children with growth hormone deficiencies. Previouslypituitary growth hormones were extracted from animal pituitary glands(sheep, beef, pig and even fish) and were used to treat human subjects.Treatment trials about 20 years ago with these hormones resulted infailure because animal growth hormones were a foreign type of proteinwhich produced neutralizing antibodies in the patients, rendering theanimal growth hormone ineffective. It is now known that only themolecular species of human growth hormone obtained from humanpituitaries can be used in human subjects.

The distinct advantages of the medium, when used in an appropriateculture procedure, are as follows: One whole human pituitary glandprovides about 2 to 3 milligrams of extractable human growth hormone.Utilizing the above medium and starting with a small single inoculum ofabout two-thirds of a human anterior pituitary gland which containsapproximately 2 milligrams of extractable growth hormone, as the initialcell culture inoculum, one achieves 10 milligrams available in the mediawithin 8 days, approximately 20 milligrams by 16 days, approximately 40milligrams by 24 days, and so on, utilizing a serial dilutionsub-culture procedure and the herein-defined nutrient media. Hence, theinitial inoculum of pituitary cells provides an amount of growth hormoneavailable in the medium for extraction and purification that isapproximately 20 times more than that present in the initial pituitarytissue which was used as the seed culture in a period of 24 days. Sincethere is a concommitant degradation of 50% of the growth hormone in themedia every three days, these values represent human growth hormoneproduced above and beyond that degraded concommitantly.

The process of the present invention is adaptable to continuousfermentation. Modifications of our present harvesting system by the useof appropriate filters to collect only the media at shorter intervalsthan 4 to 8 days, followed by recycling of the media after extraction ofthe secreted growth hormone by a continuous fermentation process wouldenable one to produce larger amounts of growth hormone.

Our experiments have confirmed that the growth hormone extracted andpurified from our process of tissue culture is biologically active,using the presently available bioassays. It has 2 units bioactivity=1 mgimmunoreactivity which is the optimum bioactivity seen with human growthhormones available at present. Another advantage of a tissue cultureprocedure for production of human growth hormone is that at least 80 to90% of the human growth hormone is in the monomer form, the simplephysiologically active form. Direct extraction of growth hormones fromhuman pituitary glands provides, in addition to monomer, dimers andpolymers; the two latter are thought to be antigenic in human subjects.Most human growth hormones available today contain monomers, dimers,polymers (approximately one-third each), the growth hormone from theScandinavian agencies (NIL-Denmark, KABI-Sweden), being better in termsof a purer monomer content than the growth hormone made by the U.S.National Pituitary Agency. It is interesting to note that the growthhormone made by our process, because of its almost pure monomericcontent, is far superior to those currently available.

At present the cost of treatment of one child with a growth hormonedeficiency is very high. Such treatment must be maintained throughoutthe entire growing span for the child. Preliminary studies by theinventors indicate that the growth hormone can be extracted from themedium utilizing minor modifications of the extraction procedures nowused for direct extraction of growth hormone from fresh gland tissues.It is estimated that the cost of preparation and extraction will not begreatly different than the processes now utilized. However, the distinctadvantage resides in the ability to obtain as much as 20 times moregrowth hormone in about three weeks time than can be obtained byextraction of one pituitary gland.

It is apparent that many modifications and variations of this inventionas hereinbefore set forth may be made without departing from the spiritand scope thereof. The specific embodiments described are given by wayof example only and the invention is limited only by the terms of theappended claims.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A nutrient culturemedium composition adapted to the production of growth hormone frompituitary cells by serial subculturing, said composition consistingessentially of effective amounts of an amino acid-rich nutrient mediumsupplemented by the addition of minor amounts of liver extract, insulin,and anti-biotic and anti-fungal agents.
 2. A composition according toclaim 1 further characterized in that the amino acid-rich medium isMedium 199-1X with Earle's modified salts.
 3. A composition according toclaim 2 further characterized in that said medium composition includesabout 5 to 20 ml of liver extract and about 6 to 20 I.U. of insulin perliter of Medium 199-1X.
 4. A composition according to claim 1 furthercharacterized in that said medium composition consists essentially ofMedium 199-1X with Earle's modified salts supplemented by about 10 mlinjectable liver extract containing B vitamins, about 10 I.U.crystalline insulin, about 500,000 units crystalline sodium penicillinG, about 500 mg streptomycin sulfate and about 50,000 units of nystatinper liter.
 5. A process for the production of growth hormone whichcomprises:(A) preparing a nutrient amino acid-rich medium compositionaccording to claim 1 in an open aeration incubation vessel, (B)dispersing cells of the anterior pituitary in said medium, (C) initiallymaintaining said cells under aerated cell growth conditions for severaldays, (D) sub-culturing the resulting initial culture and reconstitutingthe original volume with additional medium, (E) maintaining thesub-cultures under aerated cell growth conditions for an additionalseveral days, (F) serially sub-culturing every several days until thedesired optimum growth level is reached, and (G) separating the.[.media.]. .Iadd.medium .Iaddend.from the cells and extracting.Iadd.from the medium .Iaddend.the growth hormone from the sub-culturecells.
 6. A process according to claim 5 further characterized in thatsaid pituitary is human and said hormone is human growth hormone.
 7. Aprocess according to claim 5 further characterized in that the aminoacid-rich medium is Medium 199-1X with Earle's modified salts.
 8. Aprocess according to claim 7 further characterized in that said mediumcomposition includes about 5 to 20 ml of liver extract and about 6 to 20I.U. of insulin per liter of Medium 199-1X.
 9. A process according toclaim 6 further characterized in that said medium composition consistsessentially of Medium 199-1X with Earle's modified salts supplemented byabout 10 ml injectable liver extract containing B vitamins, about 10I.U. crystalline insulin, about 500,000 units crystalline sodiumpenicillin G, about 500 mg streptomycin sulfate and about 50,000 unitsof nystatin per liter.
 10. A process according to claim 6 furthercharacterized in that said initial culture is incubated for about 8days.
 11. A process according to claim 6 further characterized in thatsaid sub-cultures are incubated for about 4 to 8 days.
 12. A processaccording to claim 6 further characterized in that said initial cultureis sub-cultured at least 4 times to obtain optimal growth level.
 13. Aprocess according to claim 6 further characterized in that said culturesare aerated by a positive flow of sterile air without added carbondioxide.